/ . Biochem. 84, 65-74 (1978)
Selective Cleavage of Peptide Bonds by a Serine Protease from the Muscle Layer of Rat Small Intestine1 Keiko KOBAYASHI and Nobuhiko KATUNUMA Department of Enzyme Chemistry, Institute for Enzyme Research, School of Medicine, Tokushima University, 3-Kuramoto-cho, Tokushima, Tokushima 770 Received for publication, February 3, 1978
The kinetic constants of a serine protease from the muscle layer of rat small intestine for three ester substrates were compared with those reported for bovine chymotrypsin A. The Km values for acetyl-tyrosine ethyl ester and acetyl-phenylalanine ethyl ester were very similar to those of chymotrypsin A, but the catalytic activity per mol of serine protease was only one fiftieth as high as that of chymotrypsin A. The selectivity of action of the serine protease from the muscle layer of small intestine was examined using various polypeptide substrates, such as glucagon, oxidized insulin B chain, luteinizing hormone-releasing hormone, and neurotensin. Among the peptides bonds cleaved in these substrates, the most susceptible bonds were Tyr-Leu, Trp-Leu, Phe-Phe, Tyr-Ile, and Tyr-Gly, while Phe-Tyr and Pro-Arg-Arg-Pro were less susceptible. However, unlike the chymotrypsin group, when the amino acid on the carboxyl side of tyrosine, tryptophan or phenylalanine was serine, threonine or glutamic acid, these peptide bonds were not susceptible to the protease under the experimental conditions. These results suggest that the specificity of the serine protease from the muscle layer of small intestine is that of the chymotrypsin group, but differs from that of chymotrypsin A or C.
Previously, we reported the purification and some properties of intracellular serine proteases from various organs of rats (1-9). We obtained a homogeneous, crystalline preparation of the enzyme from the muscle layer of rat small intestine, and found that it hydrolyzed N-acetyl tyrosine ethyl
ester, a substrate of chymotrypsin, but not the substrate of trypsin, /j-toluenesulfonyl L-arginine methyl ester (1). The serine protease from small intestine also degraded milk casein, bovine hemoglobin (unpublished data) and certain enzyme proteins, such as apoornithine aminotransferase. The protease caused limited hydrolysis of these „. ,, . . substrates (2). Chemical modification of the amino 1 This work was supported by a Grant-in-Aid for Scienti. . . . „ , „. , . fie resldues of the fie Research Research from from the the Ministry Ministry of of Education, Education, Science Science t*h caitd satBS h l s t l d i n e «3*talhne protease showed and Culture of Japan (No. 137017, 1977). ' ^ ****** «*•*>« were Abbreviations: Ac, acetyl; OEt, ethyl ester; OMe, essential for the activity (1). Although various methyl ester; Bz, benzoyl; Z, benzyloxycarbonyl; Tos, properties and the primary structure of the active /Moluenesulfonyl. site were similar to those of chymotrypsin (10), Vol. 84, No. 1, 1978
65
66
K. KOBAYASHI and N. KATUNUMA
the primary structures of the amino and carboxyl terminal areas were different from those of chymotrypsin. Further, chymotrypsin did not react with the antiserum against the serine protease from small intestine in Ouchterlony double immunodiffusion test. In the present study, the kinetic constants of the enzyme for some ester substrates were compared with those reported for bovine chymotrypsin A (77, 12). To see what kind of peptide bonds in polypeptide chains were susceptible, we investigated the proteolytic actions of the serine protease from small intestine on the following polypeptide substrates : glucagon, oxidized insulin B chain, luteinizing hormone-releasing hormone, and neurotensin. The results provide fundamental information for elucidating the mechanism of degradation of certain proteins by this serine protease in vitro and in vivo. MATERIALS AND METHODS Materials—Glucagon (Sigma, crystalline No. G-4250), oxidized insulin B chain (Sigma, No. 1-2379) and the dansyl amino acids used as standards were purchased from Sigma Chem. Co. (St. Louis, Mo., U.S.A.). Dansyl chloride was obtained from SeikagakuKogyo Co., Ltd. (Tokyo). Luteinizing hormone-releasing hormone, neurotensin, and synthetic substrates were supplied from the Protein Research Foundation (Osaka). Fluorescamine was a product (Fluram) of Japan Roche Co., Ltd. (Tokyo). Dimethyl sulfoxide was obtained from Wako Pure Chemical Industries, Ltd. (Tokyo). Dowex 50W (X8) of 200-400 mesh was purchased from Muromachi Kogyo Kaisha, Ltd. (Tokyo). All other chemicals were of reagent grade. Crystalline protease was prepared from the muscle layer of the small intestine of Wistar strain rats as described previously by Katunuma et al. (7). Before use, the enzyme was crystallized with ammonium sulfate and desalted on a Sephadex G-25 column equilibrated with 0.05 M potassium phosphate buffer, pH 7.5. Hydrolysis of Ester Substrates—Two methods were employed for hydrolysis of esters. The hydrolysis of various ethyl esters was carried out by the method of Jusic et al. {13) and followed spectrophotometrically by continuous monitoring
of the increase in absorbance at 340 nm. The rate was measured at 25°C or 30°C in the presence of 0.15 M sodium pyrophosphate buffer, pH 8.0. In this case, all synthetic substrates were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 200 mg/ml. The final concentration of DMSO in the reaction mixture was 0.77%. Under the conditions used, the assay was linear with time of incubation and with the amount of enzyme added. The hydrolyses of methyl esters and some ethyl esters were assayed at 37°C by the method of Roberts (14). Reaction mixtures contained 5 /imol of synthetic substrates, 60 fimol of potassium phosphate buffer, pH 8.0, and a suitable amount of protease in a final volume of 0.5 ml. Digestion of Polypeptides—Glucagon was dissolved in 0.01 M NaOH and adjusted to a final concentration of 10 mg/ml. Other polypeptides (oxidized insulin B chain, luteinizing hormonereleasing hormone, and neurotensin) were dissolved in distilled water at a final concentration of 10 mg/ ml. The proteolytic hydrolysis was allowed to proceed at 37°C in 50 mM of potassium phosphate buffer, pH 8.0. The final concentration of each substrate in reaction mixture was 1 mg/ml, total amounts of 15-25 mg of each substrate were used. The time course of the reaction was followed by measuring increase in a-amino groups with fluorescamine by the method of Duckworth et al. (75) (excitation 390 nm, emission 475 nm). Fluorescamine was freshly prepared before determination of fluorescence. No a-amino groups were released on incubation of the individual polypeptides or protease alone, and the activity of protease remained unchanged during the digestion. Values were calculated using L-leucine as a standard and expressed as moles of leucine equivalents of aamino groups released per mol of each polypeptide as substrate. Then the mixture was diluted and acidified by adding acetic acid at a final concentration of 5 %, and rapidly frozen or applied to a Dowex SOW column. Separation of Products—Fragments of polypeptides were separated by column chromatography on Dowex 50W (X8, H + form), equilibrated with 5% acetic acid. The column (25x2.0 cm) was eluted at a flow rate of 25 ml/h under pressure and developed with a combination of gradient and stepwise increase in ammonium acetate buffer according to the method of Thompson (16) and /. Biochem.
SELECTIVE CLEAVAGE OF PEPT1DE BONDS BY A SER1NE PROTEASE
Hirs et al. (17). As the ammonium ion had to be removed from the samples before the ninhydrin reaction, the eluted fractions were hydrolyzed with alkali. For this 1 ml of 2.5 M NaOH was added to 0.2 ml samples and the mixtures were kept in an oven at 9O-95°C for 6-10 h, and then neutralized with 1.0 ml of 30% acetic acid. Then the products were subjected to the ninhydrin reaction (18). The absorption of the samples at 280 nm was also measured. The peptides isolated on Dowex 50W chromatography were lyophilized and dissolved in water. Determination of Amino-Terminal
67
Ehrlich's reagent (p-dimethylaminobenzaldehyde) as described by Easley (22). Protein Determination—Protein concentrations were determined by the method of Lowry et al. (23) using crystalline bovine serum albumin as the standard. RESULTS
Residues—
The amino-terminal residues of peptides derived from polypeptide substrates were identified as dansyl derivatives by thin layer chromatography on polyamide and/or silica gel sheets (19, 20). Amino Acid Analysis—The amino acid compositions of the products were determined by the method of Moore and Stein (21) in a Hitachi amino acid analyzer (type KLA 5). Before analysis peptide samples were hydrolyzed in a sealed tube in vacuo with 0.5-1.0 ml of 6 N HCl at 110°C for I 24 h. Peptides containing tryptophan were identified by their absorbance at 280 nm and with
Hydrolyses of Synthetic Substrates—Several synthetic substrates were used in order to determine the hydrolytic action of the serine protease from small intestine. Hydrolyses were performed at pH 8.0 by the two methods described in the " MATERIALS AND METHODS. " Experiment I was used to measure the hydrolysis of methyl and ethyl esters as substrates and experiment II was prepared for the following kinetics study. As shown in Table I, the serine protease from small intestine readily hydrolyzed N-acetyl tyrosine ethyl ester and N-acetyl phenylalanine ethyl ester, which are also good substrates for chymotrypsin, but it did not hydrolyze substrates for trypsin or elastase. The difference in the activities observed in experiments I and II was probably due to differences in
L TABLE I. Substrate specificity of a serine protease from the muscle layer of small intestine with synthetic substrates. The reaction was performed at pH 8 0 as described in the " MATERIALS AND METHODS." In experiment I the rate was determined at 37°C with 10 mM of each substrate in 0.12 M potassium phosphate buffer, pH 8.0, by the method of Roberts (14). In experiment n the rate was assayed at 30°C with 4 mg of each substrate in the presence of 0.77% of DMSO and 0.15 M sodium pyrophosphate buffer, pH 8.0, in a volume of 2.6 ml by the method of Jusic et al. (13). Experiment I Synthetic substrate Ac-Tyr-OEt Ac-Phe-OEt Ac-Trp-OEt Tyr-OEt Bz-Arg-OEt Tos-Arg-OMe Gly-OEt Lcu-OEt Ac-{Ala),-OMe Ser-OMe Val-OMe n.d.: Not determined. Vol. 84, No. 1, 1978
Experiment n
Activity (//mol/rrun/mg)
Relative activity (°/o)
• - Activity (//mol/min/mg)
Relative activity (%)
95
100
13.3 12.4
100
71 24
74.7 25.3
4.2
0.0
0.0
n.d. 0.01 n.d. 0.0 0.05 n.d. n.d.
0.0
0.0
n.d.
8 0.1 0.1
8.4 0.1 0.1
0.1
0.1
0.1
0.1
0.4
0.4
92.7 31.5 0.1 0.0 0.4
68
K. KOBAYASHI and N. KATUNUMA
reaction conditions, such as temperature, buffer and the presence or absence of DMSO. When the protease activity was assayed under the conditions of experiment H, the crystalline preparation hydrolyzed acetyl tyrosine ethyl ester at a rate of 13.33 fimol per min per mg protein; based upon a molecular weight of 25,000, this corresponds to a turnover number of 333. Kinetic Constants for the Hydrolyses of Various Substrates—The serine protease from small intestine showed similar activites as chymotrypsin with synthetic substrates, and the catalytic properties of the serine protease from small intestine and chymotrypsin were compared by measuring the kinetic parameters with three acetyl-X ethyl esters. The conditions used were essentially the same as for experiment II of Table I except that the substrate concentrations were altered and the temperature was set at 25°C. Under these conditions Lineweaver-Burk plots (not shown) gave the Km values listed in Table n , and Vmzx values for acetyl-X ethyl esters of 11.98 (X=tyrosine), 11.50 (X = phenylalanine), and 2.01 (X=tryptophan) ^mol per min per mg protein. The values of Arcat were calculated from the Vmax values and enzyme concentration, taking the molecular weight as 25,000, as described in Table II. The values of
Km, £cat, and kCatJKm for the substrates are summarized in Table II with those of bovine pancreatic chymotrypsin A for comparison (11, 12). As can be seen, the catalytic constants of the protease for these synthetic substrates were approximately one fiftieth of those of chymotrypsin A, although the present data cannot be compared exactly with the results reported by Zerner and Bender (11) and by Bender et al. (12) because conditions were slightly different. However, the Km values of this serine protease for acetyl tyrosine ethyl ester and acetyl phenylalanine ethyl ester were very similar to those of chymotrypsin A. Time Course of Hydrolysis of Glucagon by the Serine Protease—Figure 1 shows the time course of hydrolysis of glucagon. The extent of digestion by the serine protease from small intestine at 37°C was measured with fluorescamine as release of a-amino groups. With a molar ratio of 2,000 : 1, the liberation of free amino groups increased slowly with time and after 8 h approached to the value with a molar ratio of 1,000 : 1. Separation of Fragments of Glucagon Produced by the Serine Protease—Glucagon was hydrolyzed at 37°C for 8 h at a substrate to enzyme molar ratio of 1,000 : 1. Then the reaction was stopped by adding acetic acid to a final concentration of
TABLE n . Kinetic constants for the hydrolysis of substrates. Reactions were carried out at 25°C in 0.15 M of pyrophosphate buffer, pH 8.0, as described in the " MATERIALS AND METHODS." The reaction mixture contained 0.77% (v/v) DMSO to solubilize the esters. Enzyme Serine protease from small intestine
Chymotrypsin A**
Substrate (X)*
A-ct (s-1)"
Tyr Phe
6. 2 xlO" 4 1. 18x10-'
4.99 4.79
8.05 XlO3 4.06X103
Trp
4. 35x10"*
0.84
1.93x10*
Tyr
7
X10-*
193
2.76X1C
Phe
1. 2 XlO-3
173
1.44x10"
Trp
s
9
Xl0-
46.5
5.17x10*
• X of acetyl-X ethyl ester. •• Ref. (11, 12); The reaction was carried out at 25°C and pH 7.8-8.0. * The kinetic parameters shown above are defined by the equation v o = KmaxtSd/OKm+ISJ) for the process: t
»E+P
Where vo=initial velocity, Kmax—maximum velocity, [5J=initial substrate concentration, Km: Michaelis constant •=(k-i+kcadlklt E=enzyme; P=hydrolytic products. b The values for kcut were calculated from K m u /[E]. [EJ=enzyme concentration. /. Biochem.
SELECTIVE CLEAVAGE OF PEPTIDE BONDS BY A SERINE PROTEASE
69
TABLE III. Amino acid compositions of fragments obtained by digestion of glucagon with the senne protease from the muscle layer of small intestine. Experimental conditions were identical to those described in Fig. 1. Hydrolysis was carried out for 8 h at a substrate to enzyme molar ratio of 1,000:1. Total amounts of glucagon used in digestion were 5.77 ftmo\. Values in parentheses are theoretical numbers of residues in the fragment. 4
6
8
Iviolai• ratio in fragment
DIGESTION TIME (h)
Fig. 1. Hydrolysis of glucagon with the senne protease from the muscle layer of small intestine. Hydrolysis was carried out at 37°C with 1 mg/ml concentration of glucagon in 50 mM of potassium phosphate buffer, pH 8.0. Activity was determined with fluorescamine as release of ar-amino groups, as described in the " MATERIALS AND METHODS." Molar ratios of substrate to enzyme were 2,000:1 ( x ) and 1,000:1 ( • ) .
5 % and the mixture was applied to a Dowex 50W column. Three major components were isolated and subjected to N-terminal amino acid analysis by dansylation and to amino acid analysis as described in the " MATERIALS AND METHODS. " No free amino acids were detected when the components from the column were subjected to thin layer or paper chromatography without acid hydrolysis. The amino acid components and N-terminal amino acids of each fragment and their relative yields are given in Table III. The results show that the products were peptides containing residues 1-13 (fragment A), 14-25 (fragment B), and 26-29 (fragment Q, thus accounting for all the residues in glucagon. Fragment C had no absorbance at 280 nm, but fragments A and B did. Thus it was deduced that the serine protease from small intestine hydrolyzed glucagon between tyrosine (-13) and leucine (-14) and between tryptophan (-25) and leucine (-26). Thus the serine protease from small intestine may recognize the amino side of leucine. However, when short peptides containing leucine, such as Z-Ala-LeuNH,, Z-GIy-LeuNH,, and Z-Gly-Pro-Leu, were incubated at concentrations of 2 mM in 2% DMSO and 0.1 M Tris-HCl buffer, pH 8.0, for 6 h at 37°C with 20 fiu serine protease from small intestine, no liberation of ninhydrin positive substances in trichJoroacetic acid soluble fraction was detected (data not shown). Thus these results, together with those on esters, indicate that the serine proVol. 84, No. 1, 1978
Amino acid Aspartic acid Threonine Senne Glutamic acid Glycine Alanine Vahne Methionine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan Relative yield of fragment NH,-Terminal amino acid Structure ofb fragment
Fragment A
Fragment B
Fragment C
1.14 2.27 2.70 1.14 1.19
2.18 (2)
1.10 (1) 1.18 (1)
(1) (2) (3) (1)
0.94 (1) 1.92 (2)
(1)
1.07 (1) 1.03 (1) 1.26 (1) 1.71 1.28 0.65 0.92
0.82 (1) 0.90 (1)
(2) (1)
0.95 (1)
(1) (1)
1.65 (2) (I)* 35.5% 34.3% 40.6% (2.05 /imol) (1.98/itnol) Q!.34/imol) His
Leu
Leu
1-13
14-25
26-29
» See text. >> Residue numbers in the glucagon.
tease from small intestine recognizes the carboxyl side of tyrosine or tryptophan in peptide bonds of the substrate rather than the amino side of leucine. As summarized in Fig. 3, the results indicate that the chief sites of action of the serine protease from small intestine are the peptide bonds between tyrosine (-13) and leucine (-14) and between tryptophan (-25) and leucine (-26). These positions are minor sites of action of chymotrypsin C (24), but major sites of action of chymotrypsin A (25, 26). These results on susceptible sites in glucagon are
K. KOBAYASHI and N. KATUNUMA
70
consistent with those on the hydrolysis of synthetic substrates. However, it is significant that the peptide bonds between tyrosine (-10) and serine (-11) (a major site of action of chymotrypsin A, but a minor site of action of chymotrypsin C) and between phenylalanine (-6) and threonine (-7) (a major site for chymotrypsin C, but a minor site for chymotrypsin A) were not susceptible to the serine protease from small intestine under these conditions. The results show that the cleavage points of the serine protease from small intestine are more like those of chymotrypsin A than those of chymotrypsin C. Specificity Studies Using Other Polypeptides—
To obtain more information on the selectivity of the serine protease from small intestine, we examined hydrolyses of the three peptides using various molar ratios of substrate to enzyme. The time courses on hydrolysis of the polypeptides are showed in Fig. 2. The oxidized insulin B chain was rapidly digested. As shown in Fig. 2, on digestion of luteinizing hormone using a ratio of 100 : 1 for 4h, 1.0 mol of a-amino groups was released per mol of substrate. With neurotensin, the digestion took longer time to reach a plateau even at high concentration of protease (50 : 1). The fragments obtained from these three polypeptides during this incubation were separated on a Dowex 50W column. Amino acid analysis
2
4 6 8 14 DIGESTION TIME (h) Fig. 2. Degradation of three other polypeptides by the serine protease as measured by the formation of fluorescamine-reactive materials. Digestion was performed at 37°C with 1 mg/ml of each polypeptide substrate in 50 mM of potassium phosphate buffer, pH8.0, as described in the "MATERIALS AND METHODS." The molar ratios of substrate to enzyme were 500:1 (oxidized insulin B chain, x ) , 100:1 (luteinizing hormone, • ) , and 50:1 (neurotensin, A).
and dansylation of the ninhydrin-positive fractions were performed in the same way as with glucagon, and results for the three polypeptides are given in Tables IV, V, and VI. Oxidized insulin B chain was digested at a ratio of 500 : 1 at 37°C for 7 h, and then the fragments were separated on a Dowex 50W column. Three main fragments were detected. As seen from the results of amino acid analysis and dansylation of the N-terminal amino acid shown in Table IV, fragment C had two N-terminal amino TABLE IV. Amino acid compositions of fragments obtained by digestion of oxidized insulin B chain. Experimental conditions were identical to those described in Fig. 2. Hydrolysis was earned out for 7 h at a substrate to enzyme molar ratio of 500 . 1 . Total amounts of oxidized insulin B chain digested were 7.01 /imol. Values in parentheses are theoretical numbers of residues in the fragments. Molar ratio in fragment Amino acid Cysteic acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Leucine Tyrosine Phenylalanine Lysme Histidine Argmine Relative yield of fragment NHj-Terminal amino acid Structure of fragment*
Fragment
Fragment
A
B
0.90 (1)
0.94 (1) 0.97 (1)
Fragment C
1.12 (1) 0 97 (1)
1.08 (1) 2.30 (2) 1.10 (1)
2.26 (2) 0.74 (1) 1.01 (1) 1.08 (1)
1.28 1.19 1.85 2.86 0.99 1.10
(1) (1)
0.97 (1)
(2) (3) (1) (1)
0.87 (1) 0.57 (1) 0.94 (1)
1 45 (2) 1.00 (1) 30.4% 43.5% 32.9% (2.13 fi mol) (2.31 //mol) (3.05 /imoi) Phe Leu Phe (+a small amount of Tyr) 17-24
1-16
25-30 (+26-30)
Residue numbers in the oxidized insulin B chain.
/. Biochem.
SELECTIVE CLEAVAGE OF PEPTIDE BONDS BY A SERINE PROTEASE TABLE V. Amino acid compositions of fragments obtained by digestion of luteinizing hormone. Experimental conditions were identical to those described in Fig. 2. Hydrolysis was carried out for 10 h at a substrate to enzyme molar ratio of 100 : 1 . Total amounts of luteinizing hormone digested were 9.34 /imol. Values in parentheses are theoretical numbers of residues in the fragment.
TABLE VI. Amino acid compositions of fragments obtained by digestion of neurotensin. Experimental conditions were identical to those described in Fig. 2. Hydrolysis was carried out for 14 h at a substrate to enzyme molar ratio of 5 0 : 1 . Total amounts of neurotensin digested were 12.77 fimol. Values in parentheses are theoretical numbers of residues in the fragment.
Molai• ratio in fragment Amino acid
Fragment A
Fragment B
Serine Glutamic acid Proline Glycine Leucine Tyrosine Histidine Arginine
0.90 (1) 1.06 (1)
1.05 (1)
Relative yield of fragment NH,-Termina] amino acid Structure of fragment1" » See text. mone.
Fragment C
Aspartic acid Glutamic acid Proline Isoleucine Leucine Tyrosine Lysine Arginine
0.95 (1) 0.99 (1)
(I) 1
Tryptophan
b
Molar ratio in fragment Amino acid
1.17 (1) 1.83 (2) 1.02 (1) 0.96 (1) 1.07 (1)
Relative yield of fragment NH,-TerminaI amino acid Structure of fragment
9.1% 40.1% 38.5% (3.75 /jmol) (0.85 /jmol) (3.60Aimol) —
Ser
Gly
1-5
4-5
6-10
71
1
Fragment A
0.98 (1) 1.02 (1)
Fragment B
Fragment C
1.03 (1) 2.16 (2) 1.13 (1)
1.08 (1)
1.03 0.78 0.94 0.93
(1) (1) (1) (1)
0.93 (1) 0.99 (1)
51.1% 59. 5% 48.7% (6.22 pmol) ( 6.53 ftmo\) 1[7.60 (imo\) Arg
He 12-13
1-8
9-11
Residue numbers in the neurotensin.
Residue numbers in the luteinizing hor-
acids, phenylalanine and a small amount of tyrosine, but no fraction containing only phenylalanine was detected. As summarized in Fig. 3, the serine protease from small intestine mainly cleaved the peptide bonds between tyrosine (-16) and leucine (-17) and between phenylalanine (-24) and phenylalanine (-25), with slight cleavage of the bond between phenylalanine (-25) and tyrosine (-26) under these conditions. Chymotrypsin A mainly cleaved peptide bonds such as Tyr-Leu (16-17), Phe-Tyr (25-26), and Tyr-Thr (26-27) in oxidized insulin B chain, and did not split Phe-Phe (24-25) (27). Chymotrypsin C mainly cleaved Phe-Phe (24-25), Phe-Tyr (25-26), and other peptide bonds, and hydrolyzed Tyr-Leu (16-17) only slightly (24). With glucagon as substrate, the specificity of the serine protease from small intestine was more like that of chymotrypsin A than that of chymotrypsin Vol. 84, No. 1, 1978
C. These findings suggest that the serine protease from small intestine has properties in common with both chymotrypsin A and C, but also has a unique specificity. On hydrolysis of the luteinizing hormonereleasing hormone in a ratio of 100 :1 for 10 h, the peptide bond between tyrosine (-5) and glycine (-6) was the main cleavage point with slight hydrolysis of the bond between tryptophan (-3) and serine (-4), as shown in Table V. As seen from Fig. 2, the hydrolysis of neurotensin was very slow even with a high concentration of the serine protease from small intestine (substrate to enzyme ratio, 50 : 1). After digestion for 14 h, the digest was separated on a Dowex 50W column and the fragments were analyzed. Unexpectedly under these conditions the peptide bond between tyrosine and glutamic acid (3-4) in neurotensin was not cleaved but the peptide bond between arginine and arginine (8-9) was cleaved.
72
K. KOBAYASHI and N. KATLTNUMA
Glucagon 1 5 10 15 20 His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln25 Trp-Leu-Met-Asn-Thr Oxidized insulin B chain 1 5 10 15 20 Phe-Val-Asn-Gln-His-Lcu-CysSO.H-Gly-Ser-His-Leu-Val-Gln-Ala-Leu-Tyr-Leu-Val-CysSOaH-Gly-GIu-Arg25 30 Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala Luteinizing hormone-releasing hormone 1 5 10 (Pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Glyamidc
T
t
Neurotension 1 5 10 (Pyro)Glu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu
T
T
Fig. 3. Summary of the specificity of the serine protease from the muscle layer of small intestine toward glucagon, oxidized insulin B chain, luteinizing hormone-releasing hormone, and neurotensin. The unbroken arrows indicate the major sites of action of the enzyme, and broken arrows indicate other bonds cleaved by the enzyme under the individual experimental conditions employed.
The serine protease from small intestine did not hydrolyze a />-toluenesulfonyl L-arginine methyl ester, Bz-ArgOEt (Table I), Bz-ArgNHj or Z-Arg (data not shown). Although glucagon contains an Arg-Arg peptide bond, this bond was not hydrolyzed by the serine protease from small intestine under the present conditions (substrate to enzyme molar ratio, 1,000 : 1). The cleavage of the Arg-Arg bond in neurotensin is consided in the discussion. DISCUSSION In this study four polypeptides were subjected to prolonged hydrolysis with the serine protease from small intestine and then the peptides liberated were examined. Analysis of the fragments liberated from the polypeptides (Table HI-VI) showed that cleavage mainly occurred at peptide bonds containing aromatic residues. The serine protease from small intestine mainly cleaved the carboxyl side of the aromatic residues, tyrosine, tryptophan, and phenylalanine residues in polypeptides.
Although the values of fcCat for acetyl phenylalanine ethyl ester was in the same order as that for acetyl tyrosine ethyl ester and 5-times that for acetyl tryptophan ethyl ester, the carboxyl side of phenylalanine (-6) in glucagon was not cleaved by the serine protease from small intestine, while the carboxyl side of tryptophan was readily cleaved. This difference in the hydrolyses of acetyl phenylalanine ethyl ester and peptide containing phenylalanine has already been reported for elk chymotrypsin A by Lindsay and Stevenson (2*). The extent of cleavage at a particular aromatic residue may be partially dependent on the residues located on the carboxyl side of the aromatic residue. Hydrolysis of a preponderance of peptide bonds containing the carboxyl side of aromatic residues was immediately apparent, but the additional hydrolysis of the peptide bond between arginines of Pro-Arg-Arg-Pro in neurotensin was unexpected. Since the peptide bond between arginines of SerArg-Arg-Ala in glucagon was not cleaved under the experimental conditions, we consider that an Arg-Arg bond is not itself hydrolyzed. Thus the J. Biochem.
SELECTIVE CLEAVAGE OF PEPTIDE BONDS BY A SERINE PROTEASE
serine protease from small intestine may recognize peptides with proline on both sides of the Arg-Arg sequence in neurotensin. However, the serine protease from small intestine did not split the carboxyl side of all peptide bonds containing an aromatic residue. As summarized in Fig. 3, although Tyr-Leu, TrpLeu, Tyr-Ile, Tyr-Gly, and Phe-Phe are susceptible to the serine protease from small intestine, peptide bonds containing Glu, Ser, and Thr bound to the carboxyl side of an aromatic residue are not hydrolyzed, even when there is an aromatic amino acid, such as Tyr, Trp, or Phe on the amino side of these amino acids. Therefore, the requirement for two residues which form a susceptible bond appears to be very strict. This idea is supported by the fact that this serine protease from small intestine only hydrolyzed certain bonds of aromatic residues in synthetic esters. The importance of hydrophobic binding of the substrate has been discussed in relation to the actions of several mammalian enzymes including chymotrypsin (29). Many chymotrypsin-like enzymes have been reported. Human spleen cathepsin G, reported by Starkey and Barrett (30), had similar properties to serine protease. Our serine protease from the muscle layer of small intestine was also similar to cathepsin G in some properties and in its action in cleavage of the oxidized insulin B chain (31). But cathepsin G did not hydrolyze acetyl tyrosine ethyl ester and did hydrolyze the carboxyl side of leucine, so that our serine protease is different from cathepsin G. Thus our findings indicate that the catalytic properties of the serine protease from small intestine closely resemble those of bovine chymotrypsin A and porcine chymotrypsin C. However, the point of attack of the serine protease from small intestine was slightly different from those of the two chymotrypsins. Moreover the effects of various inhibitors on the serine protease from small intestine and on chymotrypsin A were different, as described by Katunuma et al. (1). The difference in action of the serine protease from small intestine and chymotrypsin may be related to a difference in structure. Recently, studies on the primary structure of the serine protease from the muscle layer of small intestine were presented by Woodbury et al. (10): of the sequence of the serine protease thus far determined, approximately Vol. 84, No. 1, 1978
73
30% of the residues are in identical loci to those in bovine chymotrypsin, trypsin and porcine elastase; however, the serine protease from small intestine seems unique in that the disulfide bond (linking residues 191 and 221) is not located in the area of the active site serine or the substrate binding pocket. The absence of a disulfide bond may be important in determining the catalytic action of the serine protease from small intestine. The biological role of the serine protease in the muscle layer of the small intestine is unknown. Previously, Kominami et al. (2-5) reported that proteases participate in degradation of ornithine aminotransferase in vitro and in vivo: hydrolysis of the apo-form of ornithine aminotransferase was limited by the serine protease from small intestine in vitro. However, the product of limited proteolysis in vivo could not be identified. The limited degradation of enzyme protein by the protease is probably due to not only the substrate specificity of the protease, but also to the susceptibility of the bonds exposed on the enzyme protein. Studies on the substrate specificity of the serine protease from the muscle layer of small intestine are useful in elucidating the reason for limited proteolysis of macromoleculc protein, as described above. In addition, since the substrate specificity of the serine protease from small intestine is stricter than that of chymotrypsin, one should be able to use the serine protease from small intestine in addition to trypsin in analysis of protein sequences. We thank Mr. H. Miyai with technical assistance in amino acid analysis. REFERENCES 1. Katunuma, N., Kominami, E., Kobayashi, K., Banno, Y., Suzuki, K , Chichibu, K., Hamaguchi, Y., & Katsunuma, T. (1975) Eur. J. Biochem. 52, 37-50 2. Kominami, E., Banno, Y., Chichibu, K., Shiotani, T., Hamaguchi, Y., & Katunuma, N. (1975) Eur. J. Biochem. 52, 51-57 3. Kominami, E. & Katunuma, N. (1976) Eur. J. Biochem. 62, 425-430 4. Katunuma, N. (1975) Rev. Physiol. Biochem. Exp. Pharmacol. 72, 83-104 5. Katunuma, N., Kominami, E., Banno, Y., Kito, K., Aoki, Y., & Urata, G. (1976) in Advances in Enzyme Regulations (Weber, G., ed.) Vol. 14, pp.
74
325-345, Pergamon Press, Oxford and New York 6. Katunuma, N. (1973) in Current Topics in Cellular Regulation (Horecker, B.L. & Stadtman, E.R., eds.) Vol. 7, pp. 175-203, Academic Press, New York 7. Katunuma, N., Katsunuma, T., Kominami, E., Suzuki, K., Hamaguchi, Y., Chichibu, K., Kobayashi, K., & Shiotani, T. (1973) in Advances in Enzyme Regulations (Weber, C , ed.) Vol. 11, pp. 37-51, Pergamon Press, Oxford and New York 8. Katunuma, N., Kominami, E., Kobayashi, K., Hamaguchi, Y., Banno, Y., Chichibu, K., Katsunuma, T., & Shiotani, T. (1975) in Intracellular Protein Turnover (Schimke, R.T. & Katunuma, N., eds.) pp. 187-204, Academic Press, New York and London 9. Katunuma, N. & Kominami, E. (1977) in Proteases in Mammalian Cells and Tissues (Barrett, A.J., ed.) pp. 151-180, EIsevier/North-Holland Biomedical Press, Amsterdam 10. Woodbury, R.G., Katunuma, N , Kobayashi, K., Titani, K., & Neurath, H. (1978) Biochemistry 17, 811-819 11. Zerner, B. & Bender, M.L. (1964) /. Am. Chem. Soc. 86, 3669-3674 12. Bender, M.L., Kezdy, F.J., & Gunther, C.R. (1964) / . Am. Chem. Soc. 86, 3714-3721 13. Jusic, M., Seifert, S., Weiss, E., Haas, R., & Heinrich, P.C. (1976) Arch. Biochem. Biophys. 177, 355-363 14. Roberts, P.S. (1958) /. Biol. Chem. 232, 285-291 15. Duckworth, W.C., Heinemann, M., & Kitabchi, A.E. (1975) Biochim. Biophys. Ada 377, 421-430 16. Thompson, A.R. (1955) Biochem. J. 61, 253-263 17. Hirs, C.H.W., Moore, S., & Stein, W.H. (1952) / . Biol. Chem. 195, 669-673
K. KOBAYASHI and N. KATUNTJMA 18. Yemm, E.W. & Cooking, E.C. (1955) Analyst 80, 209-213 19. Deyl, Z. & Rosmus, J. (1965) / . Chromatog. 20, 514-520 20. Gray, W.R. (1972) in Methods in Enzymology (Colowick, S.P. & Kaplan, N.O., eds.) Vol. 25, pp. 121-138, Academic Press, New York and London 21. Moore, S. & Stein, W.H. (1963) in Methods in Enzymology (Colowick, S.P. & Kaplan, N.O., eds.) Vol. 6, pp. 819-831, Academic Press, New York and London 22. Easley, C.W. (1965) Biochim. Biophys. Ada 107, 386-388 23. Lowry, O.H, Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) /. Biol. Chem. 193, 265-275 24. Folk, J.E. & Cole, P.W. (1965) /. Biol. Chem. 240, 193-197 25. Bromer, W.W , Sinn, L.G., & Behrens, O. (1957) /. Am. Chem. Soc. 79, 2798-2801 26. Enenkel, A.G. & Smilue, L.B. (1963) Biochemistry 2, 1449-1454 27. Sanger, F. & Tuppy, H. (1951) Biochem. J. 49, 481^90 28. Lindsay, R.M. & Stevenson, K L. (1976) Biochem. J. 155, 549-566 29. Wallace, R.A., Kurtz, A.N., & Niemann, C. (1963) Biochemistry 2, 824-836 30. Starkey, P.M. & Barrett, A.J. (1976) Biochem. J. 155, 273-278 31. Blow, A.M.J. & Barrett, A.J. (1977) Biochem. J. 161, 17-19
/ . Biochem.
Selective Cleavage of Peptide Bonds by a Serine Protease from the Muscle Layer of Rat Small Intestine1 Keiko KOBAYASHI and Nobuhiko KATUNUMA Department of Enzyme Chemistry, Institute for Enzyme Research, School of Medicine, Tokushima University, 3-Kuramoto-cho, Tokushima, Tokushima 770 Received for publication, February 3, 1978
The kinetic constants of a serine protease from the muscle layer of rat small intestine for three ester substrates were compared with those reported for bovine chymotrypsin A. The Km values for acetyl-tyrosine ethyl ester and acetyl-phenylalanine ethyl ester were very similar to those of chymotrypsin A, but the catalytic activity per mol of serine protease was only one fiftieth as high as that of chymotrypsin A. The selectivity of action of the serine protease from the muscle layer of small intestine was examined using various polypeptide substrates, such as glucagon, oxidized insulin B chain, luteinizing hormone-releasing hormone, and neurotensin. Among the peptides bonds cleaved in these substrates, the most susceptible bonds were Tyr-Leu, Trp-Leu, Phe-Phe, Tyr-Ile, and Tyr-Gly, while Phe-Tyr and Pro-Arg-Arg-Pro were less susceptible. However, unlike the chymotrypsin group, when the amino acid on the carboxyl side of tyrosine, tryptophan or phenylalanine was serine, threonine or glutamic acid, these peptide bonds were not susceptible to the protease under the experimental conditions. These results suggest that the specificity of the serine protease from the muscle layer of small intestine is that of the chymotrypsin group, but differs from that of chymotrypsin A or C.
Previously, we reported the purification and some properties of intracellular serine proteases from various organs of rats (1-9). We obtained a homogeneous, crystalline preparation of the enzyme from the muscle layer of rat small intestine, and found that it hydrolyzed N-acetyl tyrosine ethyl
ester, a substrate of chymotrypsin, but not the substrate of trypsin, /j-toluenesulfonyl L-arginine methyl ester (1). The serine protease from small intestine also degraded milk casein, bovine hemoglobin (unpublished data) and certain enzyme proteins, such as apoornithine aminotransferase. The protease caused limited hydrolysis of these „. ,, . . substrates (2). Chemical modification of the amino 1 This work was supported by a Grant-in-Aid for Scienti. . . . „ , „. , . fie resldues of the fie Research Research from from the the Ministry Ministry of of Education, Education, Science Science t*h caitd satBS h l s t l d i n e «3*talhne protease showed and Culture of Japan (No. 137017, 1977). ' ^ ****** «*•*>« were Abbreviations: Ac, acetyl; OEt, ethyl ester; OMe, essential for the activity (1). Although various methyl ester; Bz, benzoyl; Z, benzyloxycarbonyl; Tos, properties and the primary structure of the active /Moluenesulfonyl. site were similar to those of chymotrypsin (10), Vol. 84, No. 1, 1978
65
66
K. KOBAYASHI and N. KATUNUMA
the primary structures of the amino and carboxyl terminal areas were different from those of chymotrypsin. Further, chymotrypsin did not react with the antiserum against the serine protease from small intestine in Ouchterlony double immunodiffusion test. In the present study, the kinetic constants of the enzyme for some ester substrates were compared with those reported for bovine chymotrypsin A (77, 12). To see what kind of peptide bonds in polypeptide chains were susceptible, we investigated the proteolytic actions of the serine protease from small intestine on the following polypeptide substrates : glucagon, oxidized insulin B chain, luteinizing hormone-releasing hormone, and neurotensin. The results provide fundamental information for elucidating the mechanism of degradation of certain proteins by this serine protease in vitro and in vivo. MATERIALS AND METHODS Materials—Glucagon (Sigma, crystalline No. G-4250), oxidized insulin B chain (Sigma, No. 1-2379) and the dansyl amino acids used as standards were purchased from Sigma Chem. Co. (St. Louis, Mo., U.S.A.). Dansyl chloride was obtained from SeikagakuKogyo Co., Ltd. (Tokyo). Luteinizing hormone-releasing hormone, neurotensin, and synthetic substrates were supplied from the Protein Research Foundation (Osaka). Fluorescamine was a product (Fluram) of Japan Roche Co., Ltd. (Tokyo). Dimethyl sulfoxide was obtained from Wako Pure Chemical Industries, Ltd. (Tokyo). Dowex 50W (X8) of 200-400 mesh was purchased from Muromachi Kogyo Kaisha, Ltd. (Tokyo). All other chemicals were of reagent grade. Crystalline protease was prepared from the muscle layer of the small intestine of Wistar strain rats as described previously by Katunuma et al. (7). Before use, the enzyme was crystallized with ammonium sulfate and desalted on a Sephadex G-25 column equilibrated with 0.05 M potassium phosphate buffer, pH 7.5. Hydrolysis of Ester Substrates—Two methods were employed for hydrolysis of esters. The hydrolysis of various ethyl esters was carried out by the method of Jusic et al. {13) and followed spectrophotometrically by continuous monitoring
of the increase in absorbance at 340 nm. The rate was measured at 25°C or 30°C in the presence of 0.15 M sodium pyrophosphate buffer, pH 8.0. In this case, all synthetic substrates were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 200 mg/ml. The final concentration of DMSO in the reaction mixture was 0.77%. Under the conditions used, the assay was linear with time of incubation and with the amount of enzyme added. The hydrolyses of methyl esters and some ethyl esters were assayed at 37°C by the method of Roberts (14). Reaction mixtures contained 5 /imol of synthetic substrates, 60 fimol of potassium phosphate buffer, pH 8.0, and a suitable amount of protease in a final volume of 0.5 ml. Digestion of Polypeptides—Glucagon was dissolved in 0.01 M NaOH and adjusted to a final concentration of 10 mg/ml. Other polypeptides (oxidized insulin B chain, luteinizing hormonereleasing hormone, and neurotensin) were dissolved in distilled water at a final concentration of 10 mg/ ml. The proteolytic hydrolysis was allowed to proceed at 37°C in 50 mM of potassium phosphate buffer, pH 8.0. The final concentration of each substrate in reaction mixture was 1 mg/ml, total amounts of 15-25 mg of each substrate were used. The time course of the reaction was followed by measuring increase in a-amino groups with fluorescamine by the method of Duckworth et al. (75) (excitation 390 nm, emission 475 nm). Fluorescamine was freshly prepared before determination of fluorescence. No a-amino groups were released on incubation of the individual polypeptides or protease alone, and the activity of protease remained unchanged during the digestion. Values were calculated using L-leucine as a standard and expressed as moles of leucine equivalents of aamino groups released per mol of each polypeptide as substrate. Then the mixture was diluted and acidified by adding acetic acid at a final concentration of 5 %, and rapidly frozen or applied to a Dowex SOW column. Separation of Products—Fragments of polypeptides were separated by column chromatography on Dowex 50W (X8, H + form), equilibrated with 5% acetic acid. The column (25x2.0 cm) was eluted at a flow rate of 25 ml/h under pressure and developed with a combination of gradient and stepwise increase in ammonium acetate buffer according to the method of Thompson (16) and /. Biochem.
SELECTIVE CLEAVAGE OF PEPT1DE BONDS BY A SER1NE PROTEASE
Hirs et al. (17). As the ammonium ion had to be removed from the samples before the ninhydrin reaction, the eluted fractions were hydrolyzed with alkali. For this 1 ml of 2.5 M NaOH was added to 0.2 ml samples and the mixtures were kept in an oven at 9O-95°C for 6-10 h, and then neutralized with 1.0 ml of 30% acetic acid. Then the products were subjected to the ninhydrin reaction (18). The absorption of the samples at 280 nm was also measured. The peptides isolated on Dowex 50W chromatography were lyophilized and dissolved in water. Determination of Amino-Terminal
67
Ehrlich's reagent (p-dimethylaminobenzaldehyde) as described by Easley (22). Protein Determination—Protein concentrations were determined by the method of Lowry et al. (23) using crystalline bovine serum albumin as the standard. RESULTS
Residues—
The amino-terminal residues of peptides derived from polypeptide substrates were identified as dansyl derivatives by thin layer chromatography on polyamide and/or silica gel sheets (19, 20). Amino Acid Analysis—The amino acid compositions of the products were determined by the method of Moore and Stein (21) in a Hitachi amino acid analyzer (type KLA 5). Before analysis peptide samples were hydrolyzed in a sealed tube in vacuo with 0.5-1.0 ml of 6 N HCl at 110°C for I 24 h. Peptides containing tryptophan were identified by their absorbance at 280 nm and with
Hydrolyses of Synthetic Substrates—Several synthetic substrates were used in order to determine the hydrolytic action of the serine protease from small intestine. Hydrolyses were performed at pH 8.0 by the two methods described in the " MATERIALS AND METHODS. " Experiment I was used to measure the hydrolysis of methyl and ethyl esters as substrates and experiment II was prepared for the following kinetics study. As shown in Table I, the serine protease from small intestine readily hydrolyzed N-acetyl tyrosine ethyl ester and N-acetyl phenylalanine ethyl ester, which are also good substrates for chymotrypsin, but it did not hydrolyze substrates for trypsin or elastase. The difference in the activities observed in experiments I and II was probably due to differences in
L TABLE I. Substrate specificity of a serine protease from the muscle layer of small intestine with synthetic substrates. The reaction was performed at pH 8 0 as described in the " MATERIALS AND METHODS." In experiment I the rate was determined at 37°C with 10 mM of each substrate in 0.12 M potassium phosphate buffer, pH 8.0, by the method of Roberts (14). In experiment n the rate was assayed at 30°C with 4 mg of each substrate in the presence of 0.77% of DMSO and 0.15 M sodium pyrophosphate buffer, pH 8.0, in a volume of 2.6 ml by the method of Jusic et al. (13). Experiment I Synthetic substrate Ac-Tyr-OEt Ac-Phe-OEt Ac-Trp-OEt Tyr-OEt Bz-Arg-OEt Tos-Arg-OMe Gly-OEt Lcu-OEt Ac-{Ala),-OMe Ser-OMe Val-OMe n.d.: Not determined. Vol. 84, No. 1, 1978
Experiment n
Activity (//mol/rrun/mg)
Relative activity (°/o)
• - Activity (//mol/min/mg)
Relative activity (%)
95
100
13.3 12.4
100
71 24
74.7 25.3
4.2
0.0
0.0
n.d. 0.01 n.d. 0.0 0.05 n.d. n.d.
0.0
0.0
n.d.
8 0.1 0.1
8.4 0.1 0.1
0.1
0.1
0.1
0.1
0.4
0.4
92.7 31.5 0.1 0.0 0.4
68
K. KOBAYASHI and N. KATUNUMA
reaction conditions, such as temperature, buffer and the presence or absence of DMSO. When the protease activity was assayed under the conditions of experiment H, the crystalline preparation hydrolyzed acetyl tyrosine ethyl ester at a rate of 13.33 fimol per min per mg protein; based upon a molecular weight of 25,000, this corresponds to a turnover number of 333. Kinetic Constants for the Hydrolyses of Various Substrates—The serine protease from small intestine showed similar activites as chymotrypsin with synthetic substrates, and the catalytic properties of the serine protease from small intestine and chymotrypsin were compared by measuring the kinetic parameters with three acetyl-X ethyl esters. The conditions used were essentially the same as for experiment II of Table I except that the substrate concentrations were altered and the temperature was set at 25°C. Under these conditions Lineweaver-Burk plots (not shown) gave the Km values listed in Table n , and Vmzx values for acetyl-X ethyl esters of 11.98 (X=tyrosine), 11.50 (X = phenylalanine), and 2.01 (X=tryptophan) ^mol per min per mg protein. The values of Arcat were calculated from the Vmax values and enzyme concentration, taking the molecular weight as 25,000, as described in Table II. The values of
Km, £cat, and kCatJKm for the substrates are summarized in Table II with those of bovine pancreatic chymotrypsin A for comparison (11, 12). As can be seen, the catalytic constants of the protease for these synthetic substrates were approximately one fiftieth of those of chymotrypsin A, although the present data cannot be compared exactly with the results reported by Zerner and Bender (11) and by Bender et al. (12) because conditions were slightly different. However, the Km values of this serine protease for acetyl tyrosine ethyl ester and acetyl phenylalanine ethyl ester were very similar to those of chymotrypsin A. Time Course of Hydrolysis of Glucagon by the Serine Protease—Figure 1 shows the time course of hydrolysis of glucagon. The extent of digestion by the serine protease from small intestine at 37°C was measured with fluorescamine as release of a-amino groups. With a molar ratio of 2,000 : 1, the liberation of free amino groups increased slowly with time and after 8 h approached to the value with a molar ratio of 1,000 : 1. Separation of Fragments of Glucagon Produced by the Serine Protease—Glucagon was hydrolyzed at 37°C for 8 h at a substrate to enzyme molar ratio of 1,000 : 1. Then the reaction was stopped by adding acetic acid to a final concentration of
TABLE n . Kinetic constants for the hydrolysis of substrates. Reactions were carried out at 25°C in 0.15 M of pyrophosphate buffer, pH 8.0, as described in the " MATERIALS AND METHODS." The reaction mixture contained 0.77% (v/v) DMSO to solubilize the esters. Enzyme Serine protease from small intestine
Chymotrypsin A**
Substrate (X)*
A-ct (s-1)"
Tyr Phe
6. 2 xlO" 4 1. 18x10-'
4.99 4.79
8.05 XlO3 4.06X103
Trp
4. 35x10"*
0.84
1.93x10*
Tyr
7
X10-*
193
2.76X1C
Phe
1. 2 XlO-3
173
1.44x10"
Trp
s
9
Xl0-
46.5
5.17x10*
• X of acetyl-X ethyl ester. •• Ref. (11, 12); The reaction was carried out at 25°C and pH 7.8-8.0. * The kinetic parameters shown above are defined by the equation v o = KmaxtSd/OKm+ISJ) for the process: t
»E+P
Where vo=initial velocity, Kmax—maximum velocity, [5J=initial substrate concentration, Km: Michaelis constant •=(k-i+kcadlklt E=enzyme; P=hydrolytic products. b The values for kcut were calculated from K m u /[E]. [EJ=enzyme concentration. /. Biochem.
SELECTIVE CLEAVAGE OF PEPTIDE BONDS BY A SERINE PROTEASE
69
TABLE III. Amino acid compositions of fragments obtained by digestion of glucagon with the senne protease from the muscle layer of small intestine. Experimental conditions were identical to those described in Fig. 1. Hydrolysis was carried out for 8 h at a substrate to enzyme molar ratio of 1,000:1. Total amounts of glucagon used in digestion were 5.77 ftmo\. Values in parentheses are theoretical numbers of residues in the fragment. 4
6
8
Iviolai• ratio in fragment
DIGESTION TIME (h)
Fig. 1. Hydrolysis of glucagon with the senne protease from the muscle layer of small intestine. Hydrolysis was carried out at 37°C with 1 mg/ml concentration of glucagon in 50 mM of potassium phosphate buffer, pH 8.0. Activity was determined with fluorescamine as release of ar-amino groups, as described in the " MATERIALS AND METHODS." Molar ratios of substrate to enzyme were 2,000:1 ( x ) and 1,000:1 ( • ) .
5 % and the mixture was applied to a Dowex 50W column. Three major components were isolated and subjected to N-terminal amino acid analysis by dansylation and to amino acid analysis as described in the " MATERIALS AND METHODS. " No free amino acids were detected when the components from the column were subjected to thin layer or paper chromatography without acid hydrolysis. The amino acid components and N-terminal amino acids of each fragment and their relative yields are given in Table III. The results show that the products were peptides containing residues 1-13 (fragment A), 14-25 (fragment B), and 26-29 (fragment Q, thus accounting for all the residues in glucagon. Fragment C had no absorbance at 280 nm, but fragments A and B did. Thus it was deduced that the serine protease from small intestine hydrolyzed glucagon between tyrosine (-13) and leucine (-14) and between tryptophan (-25) and leucine (-26). Thus the serine protease from small intestine may recognize the amino side of leucine. However, when short peptides containing leucine, such as Z-Ala-LeuNH,, Z-GIy-LeuNH,, and Z-Gly-Pro-Leu, were incubated at concentrations of 2 mM in 2% DMSO and 0.1 M Tris-HCl buffer, pH 8.0, for 6 h at 37°C with 20 fiu serine protease from small intestine, no liberation of ninhydrin positive substances in trichJoroacetic acid soluble fraction was detected (data not shown). Thus these results, together with those on esters, indicate that the serine proVol. 84, No. 1, 1978
Amino acid Aspartic acid Threonine Senne Glutamic acid Glycine Alanine Vahne Methionine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan Relative yield of fragment NH,-Terminal amino acid Structure ofb fragment
Fragment A
Fragment B
Fragment C
1.14 2.27 2.70 1.14 1.19
2.18 (2)
1.10 (1) 1.18 (1)
(1) (2) (3) (1)
0.94 (1) 1.92 (2)
(1)
1.07 (1) 1.03 (1) 1.26 (1) 1.71 1.28 0.65 0.92
0.82 (1) 0.90 (1)
(2) (1)
0.95 (1)
(1) (1)
1.65 (2) (I)* 35.5% 34.3% 40.6% (2.05 /imol) (1.98/itnol) Q!.34/imol) His
Leu
Leu
1-13
14-25
26-29
» See text. >> Residue numbers in the glucagon.
tease from small intestine recognizes the carboxyl side of tyrosine or tryptophan in peptide bonds of the substrate rather than the amino side of leucine. As summarized in Fig. 3, the results indicate that the chief sites of action of the serine protease from small intestine are the peptide bonds between tyrosine (-13) and leucine (-14) and between tryptophan (-25) and leucine (-26). These positions are minor sites of action of chymotrypsin C (24), but major sites of action of chymotrypsin A (25, 26). These results on susceptible sites in glucagon are
K. KOBAYASHI and N. KATUNUMA
70
consistent with those on the hydrolysis of synthetic substrates. However, it is significant that the peptide bonds between tyrosine (-10) and serine (-11) (a major site of action of chymotrypsin A, but a minor site of action of chymotrypsin C) and between phenylalanine (-6) and threonine (-7) (a major site for chymotrypsin C, but a minor site for chymotrypsin A) were not susceptible to the serine protease from small intestine under these conditions. The results show that the cleavage points of the serine protease from small intestine are more like those of chymotrypsin A than those of chymotrypsin C. Specificity Studies Using Other Polypeptides—
To obtain more information on the selectivity of the serine protease from small intestine, we examined hydrolyses of the three peptides using various molar ratios of substrate to enzyme. The time courses on hydrolysis of the polypeptides are showed in Fig. 2. The oxidized insulin B chain was rapidly digested. As shown in Fig. 2, on digestion of luteinizing hormone using a ratio of 100 : 1 for 4h, 1.0 mol of a-amino groups was released per mol of substrate. With neurotensin, the digestion took longer time to reach a plateau even at high concentration of protease (50 : 1). The fragments obtained from these three polypeptides during this incubation were separated on a Dowex 50W column. Amino acid analysis
2
4 6 8 14 DIGESTION TIME (h) Fig. 2. Degradation of three other polypeptides by the serine protease as measured by the formation of fluorescamine-reactive materials. Digestion was performed at 37°C with 1 mg/ml of each polypeptide substrate in 50 mM of potassium phosphate buffer, pH8.0, as described in the "MATERIALS AND METHODS." The molar ratios of substrate to enzyme were 500:1 (oxidized insulin B chain, x ) , 100:1 (luteinizing hormone, • ) , and 50:1 (neurotensin, A).
and dansylation of the ninhydrin-positive fractions were performed in the same way as with glucagon, and results for the three polypeptides are given in Tables IV, V, and VI. Oxidized insulin B chain was digested at a ratio of 500 : 1 at 37°C for 7 h, and then the fragments were separated on a Dowex 50W column. Three main fragments were detected. As seen from the results of amino acid analysis and dansylation of the N-terminal amino acid shown in Table IV, fragment C had two N-terminal amino TABLE IV. Amino acid compositions of fragments obtained by digestion of oxidized insulin B chain. Experimental conditions were identical to those described in Fig. 2. Hydrolysis was earned out for 7 h at a substrate to enzyme molar ratio of 500 . 1 . Total amounts of oxidized insulin B chain digested were 7.01 /imol. Values in parentheses are theoretical numbers of residues in the fragments. Molar ratio in fragment Amino acid Cysteic acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Leucine Tyrosine Phenylalanine Lysme Histidine Argmine Relative yield of fragment NHj-Terminal amino acid Structure of fragment*
Fragment
Fragment
A
B
0.90 (1)
0.94 (1) 0.97 (1)
Fragment C
1.12 (1) 0 97 (1)
1.08 (1) 2.30 (2) 1.10 (1)
2.26 (2) 0.74 (1) 1.01 (1) 1.08 (1)
1.28 1.19 1.85 2.86 0.99 1.10
(1) (1)
0.97 (1)
(2) (3) (1) (1)
0.87 (1) 0.57 (1) 0.94 (1)
1 45 (2) 1.00 (1) 30.4% 43.5% 32.9% (2.13 fi mol) (2.31 //mol) (3.05 /imoi) Phe Leu Phe (+a small amount of Tyr) 17-24
1-16
25-30 (+26-30)
Residue numbers in the oxidized insulin B chain.
/. Biochem.
SELECTIVE CLEAVAGE OF PEPTIDE BONDS BY A SERINE PROTEASE TABLE V. Amino acid compositions of fragments obtained by digestion of luteinizing hormone. Experimental conditions were identical to those described in Fig. 2. Hydrolysis was carried out for 10 h at a substrate to enzyme molar ratio of 100 : 1 . Total amounts of luteinizing hormone digested were 9.34 /imol. Values in parentheses are theoretical numbers of residues in the fragment.
TABLE VI. Amino acid compositions of fragments obtained by digestion of neurotensin. Experimental conditions were identical to those described in Fig. 2. Hydrolysis was carried out for 14 h at a substrate to enzyme molar ratio of 5 0 : 1 . Total amounts of neurotensin digested were 12.77 fimol. Values in parentheses are theoretical numbers of residues in the fragment.
Molai• ratio in fragment Amino acid
Fragment A
Fragment B
Serine Glutamic acid Proline Glycine Leucine Tyrosine Histidine Arginine
0.90 (1) 1.06 (1)
1.05 (1)
Relative yield of fragment NH,-Termina] amino acid Structure of fragment1" » See text. mone.
Fragment C
Aspartic acid Glutamic acid Proline Isoleucine Leucine Tyrosine Lysine Arginine
0.95 (1) 0.99 (1)
(I) 1
Tryptophan
b
Molar ratio in fragment Amino acid
1.17 (1) 1.83 (2) 1.02 (1) 0.96 (1) 1.07 (1)
Relative yield of fragment NH,-TerminaI amino acid Structure of fragment
9.1% 40.1% 38.5% (3.75 /jmol) (0.85 /jmol) (3.60Aimol) —
Ser
Gly
1-5
4-5
6-10
71
1
Fragment A
0.98 (1) 1.02 (1)
Fragment B
Fragment C
1.03 (1) 2.16 (2) 1.13 (1)
1.08 (1)
1.03 0.78 0.94 0.93
(1) (1) (1) (1)
0.93 (1) 0.99 (1)
51.1% 59. 5% 48.7% (6.22 pmol) ( 6.53 ftmo\) 1[7.60 (imo\) Arg
He 12-13
1-8
9-11
Residue numbers in the neurotensin.
Residue numbers in the luteinizing hor-
acids, phenylalanine and a small amount of tyrosine, but no fraction containing only phenylalanine was detected. As summarized in Fig. 3, the serine protease from small intestine mainly cleaved the peptide bonds between tyrosine (-16) and leucine (-17) and between phenylalanine (-24) and phenylalanine (-25), with slight cleavage of the bond between phenylalanine (-25) and tyrosine (-26) under these conditions. Chymotrypsin A mainly cleaved peptide bonds such as Tyr-Leu (16-17), Phe-Tyr (25-26), and Tyr-Thr (26-27) in oxidized insulin B chain, and did not split Phe-Phe (24-25) (27). Chymotrypsin C mainly cleaved Phe-Phe (24-25), Phe-Tyr (25-26), and other peptide bonds, and hydrolyzed Tyr-Leu (16-17) only slightly (24). With glucagon as substrate, the specificity of the serine protease from small intestine was more like that of chymotrypsin A than that of chymotrypsin Vol. 84, No. 1, 1978
C. These findings suggest that the serine protease from small intestine has properties in common with both chymotrypsin A and C, but also has a unique specificity. On hydrolysis of the luteinizing hormonereleasing hormone in a ratio of 100 :1 for 10 h, the peptide bond between tyrosine (-5) and glycine (-6) was the main cleavage point with slight hydrolysis of the bond between tryptophan (-3) and serine (-4), as shown in Table V. As seen from Fig. 2, the hydrolysis of neurotensin was very slow even with a high concentration of the serine protease from small intestine (substrate to enzyme ratio, 50 : 1). After digestion for 14 h, the digest was separated on a Dowex 50W column and the fragments were analyzed. Unexpectedly under these conditions the peptide bond between tyrosine and glutamic acid (3-4) in neurotensin was not cleaved but the peptide bond between arginine and arginine (8-9) was cleaved.
72
K. KOBAYASHI and N. KATLTNUMA
Glucagon 1 5 10 15 20 His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln25 Trp-Leu-Met-Asn-Thr Oxidized insulin B chain 1 5 10 15 20 Phe-Val-Asn-Gln-His-Lcu-CysSO.H-Gly-Ser-His-Leu-Val-Gln-Ala-Leu-Tyr-Leu-Val-CysSOaH-Gly-GIu-Arg25 30 Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala Luteinizing hormone-releasing hormone 1 5 10 (Pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Glyamidc
T
t
Neurotension 1 5 10 (Pyro)Glu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu
T
T
Fig. 3. Summary of the specificity of the serine protease from the muscle layer of small intestine toward glucagon, oxidized insulin B chain, luteinizing hormone-releasing hormone, and neurotensin. The unbroken arrows indicate the major sites of action of the enzyme, and broken arrows indicate other bonds cleaved by the enzyme under the individual experimental conditions employed.
The serine protease from small intestine did not hydrolyze a />-toluenesulfonyl L-arginine methyl ester, Bz-ArgOEt (Table I), Bz-ArgNHj or Z-Arg (data not shown). Although glucagon contains an Arg-Arg peptide bond, this bond was not hydrolyzed by the serine protease from small intestine under the present conditions (substrate to enzyme molar ratio, 1,000 : 1). The cleavage of the Arg-Arg bond in neurotensin is consided in the discussion. DISCUSSION In this study four polypeptides were subjected to prolonged hydrolysis with the serine protease from small intestine and then the peptides liberated were examined. Analysis of the fragments liberated from the polypeptides (Table HI-VI) showed that cleavage mainly occurred at peptide bonds containing aromatic residues. The serine protease from small intestine mainly cleaved the carboxyl side of the aromatic residues, tyrosine, tryptophan, and phenylalanine residues in polypeptides.
Although the values of fcCat for acetyl phenylalanine ethyl ester was in the same order as that for acetyl tyrosine ethyl ester and 5-times that for acetyl tryptophan ethyl ester, the carboxyl side of phenylalanine (-6) in glucagon was not cleaved by the serine protease from small intestine, while the carboxyl side of tryptophan was readily cleaved. This difference in the hydrolyses of acetyl phenylalanine ethyl ester and peptide containing phenylalanine has already been reported for elk chymotrypsin A by Lindsay and Stevenson (2*). The extent of cleavage at a particular aromatic residue may be partially dependent on the residues located on the carboxyl side of the aromatic residue. Hydrolysis of a preponderance of peptide bonds containing the carboxyl side of aromatic residues was immediately apparent, but the additional hydrolysis of the peptide bond between arginines of Pro-Arg-Arg-Pro in neurotensin was unexpected. Since the peptide bond between arginines of SerArg-Arg-Ala in glucagon was not cleaved under the experimental conditions, we consider that an Arg-Arg bond is not itself hydrolyzed. Thus the J. Biochem.
SELECTIVE CLEAVAGE OF PEPTIDE BONDS BY A SERINE PROTEASE
serine protease from small intestine may recognize peptides with proline on both sides of the Arg-Arg sequence in neurotensin. However, the serine protease from small intestine did not split the carboxyl side of all peptide bonds containing an aromatic residue. As summarized in Fig. 3, although Tyr-Leu, TrpLeu, Tyr-Ile, Tyr-Gly, and Phe-Phe are susceptible to the serine protease from small intestine, peptide bonds containing Glu, Ser, and Thr bound to the carboxyl side of an aromatic residue are not hydrolyzed, even when there is an aromatic amino acid, such as Tyr, Trp, or Phe on the amino side of these amino acids. Therefore, the requirement for two residues which form a susceptible bond appears to be very strict. This idea is supported by the fact that this serine protease from small intestine only hydrolyzed certain bonds of aromatic residues in synthetic esters. The importance of hydrophobic binding of the substrate has been discussed in relation to the actions of several mammalian enzymes including chymotrypsin (29). Many chymotrypsin-like enzymes have been reported. Human spleen cathepsin G, reported by Starkey and Barrett (30), had similar properties to serine protease. Our serine protease from the muscle layer of small intestine was also similar to cathepsin G in some properties and in its action in cleavage of the oxidized insulin B chain (31). But cathepsin G did not hydrolyze acetyl tyrosine ethyl ester and did hydrolyze the carboxyl side of leucine, so that our serine protease is different from cathepsin G. Thus our findings indicate that the catalytic properties of the serine protease from small intestine closely resemble those of bovine chymotrypsin A and porcine chymotrypsin C. However, the point of attack of the serine protease from small intestine was slightly different from those of the two chymotrypsins. Moreover the effects of various inhibitors on the serine protease from small intestine and on chymotrypsin A were different, as described by Katunuma et al. (1). The difference in action of the serine protease from small intestine and chymotrypsin may be related to a difference in structure. Recently, studies on the primary structure of the serine protease from the muscle layer of small intestine were presented by Woodbury et al. (10): of the sequence of the serine protease thus far determined, approximately Vol. 84, No. 1, 1978
73
30% of the residues are in identical loci to those in bovine chymotrypsin, trypsin and porcine elastase; however, the serine protease from small intestine seems unique in that the disulfide bond (linking residues 191 and 221) is not located in the area of the active site serine or the substrate binding pocket. The absence of a disulfide bond may be important in determining the catalytic action of the serine protease from small intestine. The biological role of the serine protease in the muscle layer of the small intestine is unknown. Previously, Kominami et al. (2-5) reported that proteases participate in degradation of ornithine aminotransferase in vitro and in vivo: hydrolysis of the apo-form of ornithine aminotransferase was limited by the serine protease from small intestine in vitro. However, the product of limited proteolysis in vivo could not be identified. The limited degradation of enzyme protein by the protease is probably due to not only the substrate specificity of the protease, but also to the susceptibility of the bonds exposed on the enzyme protein. Studies on the substrate specificity of the serine protease from the muscle layer of small intestine are useful in elucidating the reason for limited proteolysis of macromoleculc protein, as described above. In addition, since the substrate specificity of the serine protease from small intestine is stricter than that of chymotrypsin, one should be able to use the serine protease from small intestine in addition to trypsin in analysis of protein sequences. We thank Mr. H. Miyai with technical assistance in amino acid analysis. REFERENCES 1. Katunuma, N., Kominami, E., Kobayashi, K., Banno, Y., Suzuki, K , Chichibu, K., Hamaguchi, Y., & Katsunuma, T. (1975) Eur. J. Biochem. 52, 37-50 2. Kominami, E., Banno, Y., Chichibu, K., Shiotani, T., Hamaguchi, Y., & Katunuma, N. (1975) Eur. J. Biochem. 52, 51-57 3. Kominami, E. & Katunuma, N. (1976) Eur. J. Biochem. 62, 425-430 4. Katunuma, N. (1975) Rev. Physiol. Biochem. Exp. Pharmacol. 72, 83-104 5. Katunuma, N., Kominami, E., Banno, Y., Kito, K., Aoki, Y., & Urata, G. (1976) in Advances in Enzyme Regulations (Weber, G., ed.) Vol. 14, pp.
74
325-345, Pergamon Press, Oxford and New York 6. Katunuma, N. (1973) in Current Topics in Cellular Regulation (Horecker, B.L. & Stadtman, E.R., eds.) Vol. 7, pp. 175-203, Academic Press, New York 7. Katunuma, N., Katsunuma, T., Kominami, E., Suzuki, K., Hamaguchi, Y., Chichibu, K., Kobayashi, K., & Shiotani, T. (1973) in Advances in Enzyme Regulations (Weber, C , ed.) Vol. 11, pp. 37-51, Pergamon Press, Oxford and New York 8. Katunuma, N., Kominami, E., Kobayashi, K., Hamaguchi, Y., Banno, Y., Chichibu, K., Katsunuma, T., & Shiotani, T. (1975) in Intracellular Protein Turnover (Schimke, R.T. & Katunuma, N., eds.) pp. 187-204, Academic Press, New York and London 9. Katunuma, N. & Kominami, E. (1977) in Proteases in Mammalian Cells and Tissues (Barrett, A.J., ed.) pp. 151-180, EIsevier/North-Holland Biomedical Press, Amsterdam 10. Woodbury, R.G., Katunuma, N , Kobayashi, K., Titani, K., & Neurath, H. (1978) Biochemistry 17, 811-819 11. Zerner, B. & Bender, M.L. (1964) /. Am. Chem. Soc. 86, 3669-3674 12. Bender, M.L., Kezdy, F.J., & Gunther, C.R. (1964) / . Am. Chem. Soc. 86, 3714-3721 13. Jusic, M., Seifert, S., Weiss, E., Haas, R., & Heinrich, P.C. (1976) Arch. Biochem. Biophys. 177, 355-363 14. Roberts, P.S. (1958) /. Biol. Chem. 232, 285-291 15. Duckworth, W.C., Heinemann, M., & Kitabchi, A.E. (1975) Biochim. Biophys. Ada 377, 421-430 16. Thompson, A.R. (1955) Biochem. J. 61, 253-263 17. Hirs, C.H.W., Moore, S., & Stein, W.H. (1952) / . Biol. Chem. 195, 669-673
K. KOBAYASHI and N. KATUNTJMA 18. Yemm, E.W. & Cooking, E.C. (1955) Analyst 80, 209-213 19. Deyl, Z. & Rosmus, J. (1965) / . Chromatog. 20, 514-520 20. Gray, W.R. (1972) in Methods in Enzymology (Colowick, S.P. & Kaplan, N.O., eds.) Vol. 25, pp. 121-138, Academic Press, New York and London 21. Moore, S. & Stein, W.H. (1963) in Methods in Enzymology (Colowick, S.P. & Kaplan, N.O., eds.) Vol. 6, pp. 819-831, Academic Press, New York and London 22. Easley, C.W. (1965) Biochim. Biophys. Ada 107, 386-388 23. Lowry, O.H, Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) /. Biol. Chem. 193, 265-275 24. Folk, J.E. & Cole, P.W. (1965) /. Biol. Chem. 240, 193-197 25. Bromer, W.W , Sinn, L.G., & Behrens, O. (1957) /. Am. Chem. Soc. 79, 2798-2801 26. Enenkel, A.G. & Smilue, L.B. (1963) Biochemistry 2, 1449-1454 27. Sanger, F. & Tuppy, H. (1951) Biochem. J. 49, 481^90 28. Lindsay, R.M. & Stevenson, K L. (1976) Biochem. J. 155, 549-566 29. Wallace, R.A., Kurtz, A.N., & Niemann, C. (1963) Biochemistry 2, 824-836 30. Starkey, P.M. & Barrett, A.J. (1976) Biochem. J. 155, 273-278 31. Blow, A.M.J. & Barrett, A.J. (1977) Biochem. J. 161, 17-19
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